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The Avr4 effector, secreted by <i>Cladosporium fulvum</i>, has been demonstrated to be involved in pathogen virulence. Recent study further demonstrated that Avr4 is highly conserved among several Cercospora species, indicating a potential important role of this gene in the fungus virulence. Therefore, further investigation to determine whether this fungal effector gene is present in <i>Cercospora kikuchii</i>, the causal agent of soybean cercospora leaf blight (CLB) disease, and whether it plays any role in CLB development, is of great interest. These studies can provide important information in understanding host-pathogen interaction and in developing new approaches to enhance resistance to <i>C. kikuchii</i> infection in soybean. In the present study, we have cloned the <i>avr4</i> gene from <i>C. kikuchii</i>, and mutants lacking the expression of <i>avr4</i> have been created by homologous recombination using fusion PCR to generate a construct containing the hygromycin cassette fused with <i>avr4</i> 5′ and 3′ fragments. Virulence assays using soybean leaves revealed that the <i>avr4</i> mutants have reduced fungal growth. Furthermore, disruption of the <i>avr4</i> gene resulted in mutants with significant reduced cercosporin toxin biosynthesis (CTB) and CTB genes expression. The results indicate that in addition to the previous reported chitin binding activity to protect pathogen during infection, Avr4 also contributes to the virulence of <i>Cercospora kikuchii</i> on soybean through regulation of cercosporin biosynthesis.